The homeodomain is the highly conserved DNA-binding domain of a class of proteins that function as transcriptional regulators, specifying positional and temporal information in the commitment of embryonic cells to specific developmental pathways. An early step in the cascade of events associated with development is the sequence-specific binding of the transcriptional regulator (the homeodomain-containing protein) to a specific sequence or a specific set of sequences of DNA. The homeodomain studied in the last four years and published in Biochemistry, 2001 is encoded by the vnd (ventral nervous system defective)/NK-2 gene of Drosophila melanogaster and is the parent member of the vnd/NK-2 class of homeodomains first described by Kim & Nirenberg. [Studies on the NKX-2.5 homeodomain protein, an essential transcriptional regulator in the early human heart development, continue but results remain preliminary and are not reported here.] The conformational stabilities of the vnd/NK-2 homeodomain [HD(wt); residues 1-80 that encompasses the 60 residue homeodomain] and those harboring mutations in helix III of the DNA recognition site [HD(H52R) and HD(H52R/T56W)] have been investigated by differential scanning calorimetry (DSC) and ellipticity changes at 222 nm. Thermal unfolding reactions at pH 7.4 are reversible and repeatable in the presence of 50-500 mM NaCl with delta Cp = 0.52 +/- 0.04 kcal/K mol. A substantial stabilization of HD(wt) is produced by 50 mM phosphate or by the addition of 100-500 mM NaCl to 50 mM Hepes pH 7.4 buffer (from a transition temperature of 35.5 to 43 - 51 C with vant Hoff enthalpy changes of 47 +/- 5 kcal/mol). The order of stability is HD(H52R/T56W) > HD(H52R) > HD(wt), irrespective of the anions present. Progress curves for ellipticity changes at 222 nm as a function of increasing temperature are fitted well by a two-state unfolding model, and the cooperativity of secondary structure changes is greater for mutant homeodomains than for HD(wt) and also is increased by adding 100 mM NaCl to Hepes buffer. A 33% quench of the intrinsic tryptophanyl residue fluorescence of HD(wt) by phosphate binding (with a dissociation constant of ca. 2.6 mM estimated for phosphate) is reversed 60% by DNA binding. Thermodynamic parameters for vnd/NK-2 homeodomain proteins binding sequence-specific 18 bp DNA have been determined by isothermal titration calorimetry (10 - 30 C). Values of heat capacity changes are +0.25, -0.17, and -0.10 +/- 0.04 kcal/K mol for HD(wt), HD(H52R), and HD(H52R/T56W) binding duplex DNA, respectively. Interactions of homeodomains with DNA are enthalpically controlled at 298 K and pH 7.4 with corresponding enthalpy changes of -6.6 +/- 0.5, -10.8 +/- 0.1, and -9.0 +/-0.6 kcal/mol and Gibbs free energy changes of -11.0 +/- 0.1, -11.0 +/- 0.1, and -11.3 +/- 0.3 kcal/mol with a binding stoichiometry of 1.0 +/- 0.1. Thermodynamic parameters for DNA binding are not predicted from homeodomain structural changes that occur upon complexing to DNA and therefore must reflect also solvent and possibly DNA rearrangements.